This invention relates to the field of electrophoresis, more specifically, to a sieving medium for electrophoretic separation of biopolymers.
Electrophoresis is conducted in free solution or within a support medium which serves to minimize convection and diffusion, and in many cases to effect molecular sieving (see Andrews, Electrophoresis, 2nd Edition, Clarendon Press, Oxford, 1986). Cellulose acetate and paper are examples of electrophoresis matrices which do not affect separation, but serve primarily as anti-convective supports. Polyacrylamide and agarose gels have been used because they not only minimize convection and diffusion, but also act as molecular sieving matrices to molecules of a size comparable to that of the gel pores.
Polyacrylamide gels are typically prepared by free-radical polymerization of acrylamide in the presence of a crosslinking monomer, typically N,N'-methylene bisacrylamide. Agarose gels are prepared by dissolving agarose in a buffer solution at high temperature (70.degree.-96.degree. C., depending on the grade of agarose), and then allowing the solution to cool whereby an elastic gel is formed. Other forms of media have been described.
Numerous alternative acrylamide monomers and crosslinkers have been proposed with the objective of improving some aspect of gel performance, but leaving the basic procedure of gel preparation unaltered. (See general review by Righetti et al, J. Chromatography, 638, 165 (1993)). U.S. Pat. No. 5,055,517 to Shorr et al. discloses gels formed by polymerizing one or more acrylamide monomers, including various N-substituted acrylamide derivatives, with crosslinkers based on ethylene glycol dimethacrylate or poly (ethylene glycol) dimethacrylate. Mori et al in U.S. Pat. No. 5,164,057 describe electrophoresis media comprising at least one water-insolubilized temperature-responsive polymeric compound having an LCST. Mori et al describe conventional methods for rendering the matrix water-insolubilized, such as casting a crosslinked, macroscopic gel slab.
Radola et al, Biochim Biophys. Acta, 386, 181 (1974) describe isoelectric focusing in a flat bed of granulated, hydrophilic gel beads, such as dextran or polyacrylamide. In this method, a slurry of the granulated gel is prepared with excess water so that the slurry viscosity is low enough to allow it to be poured into a flat tray. After pouring, the excess water must be removed prior to conducting the separation. This is done either by evaporation or by transferring water into an absorbing material from the ends of the tray. The requirement of drying the bed after pouring adds significant time to the preparation, and is a source of inconsistent and poor separations due to insufficient or excess removal of solution after pouring. In addition, granulated beds have not been used for electrophoretic separations requiring molecular sieving, and electrophoretic migration is believed to occur in the interstices remaining between the granular particles of such conventional beds.
Native agarose is used but typically not at concentrations greater than 5% due the high viscosity of such solutions and the corresponding difficulty of pouring. Various agarose derivatives have been disclosed which have a finer pore structure, and therefore higher sieving power for small molecules, than native agarose. An example is hydroxyethylated agarose, described by Guiseley in U.S. Pat. No. 3,956,273. Nochumson et al (U.S. Pat. No. 5,143,646) describe small-pore resolving agarose gel blends comprising at least one depolymerized agarose.
Peacock and Dingman, Biochemistry, 7(21)668 (1968) disclose the preparation and use of agarose-acrylamide composite gels for electrophoresis. Polyacrylamide gels of very low concentration, which may be desirable to fractionate large molecules, do not have sufficient strength for handling. By polymerizing the acrylamide and crosslinker in the presence of agarose (which can be gelled before or after polymerizing the acrylamide), a composite matrix is created whereby the agarose provides structural support and the polyacrylamide acts as the primary sieving matrix. Agarose combined with prepolymerized, uncrosslinked polyacrylamide has also been suggested as an electrophoresis medium (Bode, Analytical Biochemistry, 83,204, 1977). However, this approach has disadvantages including the increased viscosity at the pouring temperature due to the presence of the linear polyacrylamide, and the potential for extraction of the unbound, water-soluble polymer during electrophoresis (particularly during submerged gel electrophoresis.)
As an alternative to covalently crosslinked gels, solutions of uncrosslinked, entangled polymers, such as polyethylene glycol, polyacrylamide, dextran, polyethylene oxide, methylcellulose, hydroxypropylmethylcellulose, and hydroxyethylcellulose have been employed as electrophoresis media (Bode, FEBS Lett., 65, 56, 1976; Tietz et al, Electrophoresis, 7, 217, 1986; Grossman and Soane, Biopolymers, 31, 1221, 1991; P. D. Grossman and D. S. Soane, J. Chromatography, 559, 257 (1991); M. J. Bode, FEBS Lett., 65, 56 (1991); M. Zhu, D. L. Hansen, S. Burd and F. Gannon, J. chroniatography, 480, 311 (1989); A. M. Chin and J. C. Colburn, Am. Biotech. Lab., 7, 16 (1989); D. Tietz, M. M. Gottlieb, J. S. Fawcett and A. Chrambach, Electrophoresis, 7, 217 (1986)). An advantage of this approach is that the medium can be injected into and flushed from a separation channel, such as a micro-capillary. Shortcomings of this approach include lower resolution than typically observed for non-viscous, crosslinked polyacrylamide matrices, and the often high solution viscosities, required. Osterhoudt et al (U.S. Pat. No. 5,149,419) describe preformed, water-soluble, acrylamide based copolymers comprising a minor proportion of a comonomer containing a crosslinking site. This crosslinking site is used to convert the polymer solution into a crosslinked electrophoresis network in situ by a reaction that does not involve free-radical addition. The crosslinking reaction described be Osterhoudt et al is not believed to be reversible, i.e., the gel network is apparently not reconvertible to a polymer solution.
The present invention is based on the discovery of an induced, and reversible, viscosity change, which changes the sieving characteristics of a polymer matrix. It is well known that the solubility of polymers in aqueous solution depends on several solution conditions including temperature, pH, ionic strength, the specific ions present in solution, and the presence and concentration of other molecular components in solution including other polymers, cosolvents, surfactants, etc. It was disclosed, for example, by Heskins and Guillet (J. Macromol. Sci.--Chem., A2 (8), 1441, 1968) that aqueous solutions of poly(N-isopropylacrylamide) exhibit a lower critical solution temperature (LCST), i.e. that aqueous solutions of this polymer phase separate at temperatures above the LCST. Taylor and Cerankowski (J. Polym. Sci., Polym. Chem. Ed., 13, 2551, 1975) discuss numerous polymers that exhibit LCST behavior in aqueous solutions, and they demonstrate the change in swelling of a polymer film in solution that occurs at the LCST.
It is generally recognized that if a polymer exhibits an LCST in aqueous solution, then a crosslinked network of this polymer solvated in the same solution will undergo a significant (i.e., greater than 50%) increase in swelling, or volume upon a temperature decrease from above to below the LCST. This has been demonstrated for poly(N-isopropylacrylamide) (Hirokawa and Tanaka, J. Chem. Phys., 81, 6379, 1984) and for poly(N,N'-diethylacrylamide) (Ilavsky et al, Polym. Bull., 7, 107, 1982).
It is further recognized that any number of solution variables (e.g. pH, ionic strength, cosolvent concentration) which affect the solubility of a polymer in solution will also affect the solution swelling characteristics of a crosslinked gel composed primarily of the polymer. For example, the solubility of uncrosslinked polyacrylamide in solutions of acetone and water is known to decrease as the concentration of acetone is increased, and crosslinked polyacrylamide gel is known to exhibit a significant decrease in swelling in solutions of acetone and water as the acetone concentration is increased.
The swelling and contraction of polyacrylamide gel slabs in aqueous solutions due to temperature, various solutes and salts was investigated by Boyde (J. Chrom., 124, 219 1976). Polyacrylamide is highly soluble in water and does not exhibit an LCST. Thus, while the swelling of polyacrylamide gel slabs in water was shown by Boyde to vary up to 40% with temperature, large (i.e., greater than 50 or 100%) changes in gel volume were not observed in response to temperature changes.
Tanaka et al (U.S. Pat. No. 5,100,933) disclose a method of causing a discontinuous volume change in a gel in response to changes in metal ion concentration, whereby the gel is an ionized, crosslinked polyacrylamide. In U.S. Pat. No. 4,732,930, Tanaka et al disclose ionic isopropylacrylmide gels which exhibit volume changes in response to solvent composition, temperature, pH or ion composition.
The present invention is useful for electrophoresis, in general, and, in particular, for improving capillary electrophoresis (CE), which has found widespread applications in analytical and biomedical research. The scope and sophistication of CE are rapidly increasing. CE can perform analytical separations that are often substantially better than those using established chromatographic methods such as high-performance liquid chromatography (HPLC). The separation modes of the conventional electrophoretic methods are slow, labor-intensive, prone to relatively poor reproducibility and have limited quantitative capability. Furthermore, it has been difficult to fully automate the process. The major advantages of capillary electrophoresis are that it can be fully automated, offers high resolution, and can quantitate minute amounts of samples, as reviewed by N. A. Guzman, L. Hernandez and S. Terabe, Analytical Biotechnology, Chapter 1, ed. by C. Horvath and J. G. Nikelly. ACS symposium series, ACS, Washington, DC (1990). These capabilities lie far beyond those of traditional electrophoretic methods.
CE has recently been used in the analysis of an extremely wide variety of molecules, including organic and inorganic anions and cations, drugs, dyes and their precursors, vitamins, carbohydrates, catecholamines, amino acids, proteins and peptides, nucleic acids, nucleotides, DNA and oligonucleotides. In comparison with gas chromatography, supercritical fluid chromatography, and liquid chromatography, CE is the best separation technique from the point of view of molecular weight range of applicability. It is possible to separate in the same column species ranging in size from free amino acids to large proteins associated with complex molecular matrices.
From the detection point of view, HPLC provides better concentration sensitivity and CE provides better mass sensitivity. However, initial attempts to resolve complex mixtures of biological macromolecules in open CE columns were disappointing. The complex protein macromolecules present a serious problem when using untreated fused-silica capillaries due to the adsorption of many proteins onto the walls of the capillary. With oligonucleotides, the unfavorable mass-to-charge ratio tends to cause comigration of larger mixture components.
A highly advantageous solution to these difficulties was the development of gel-filled capillaries. Remarkably high separation efficiency has been obtained by gel-filled CE. To accomplish size selection in electrophoretic separation of mixtures of nucleic acids and SDS-denatured proteins, a cross-linked gel matrix is employed. However, the routine preparation of homogeneous stress-free gels in capillaries is difficult due to polymerization induced shrinkage and appearance of bubbles inside the capillaries.
The resolving power of capillary electrophoresis (CE) using the prior art entangled polymer solutions as the separation media is not good for large analyte molecules, presumably as a result of the relevant time scales of the sieving polymers and analyte molecules. The residence time (or passage time) of analyte molecule in a mesh is controlled by the size and electrophoretic mobility of the analyte, mesh size of the network, and the imposed electric field strength. The life time of entanglement, i.e., mobility of strands forming the mesh, depends on network integrity, the length and concentration of macromolecules constituting the network. In order to achieve good resolution, the relaxation time of the entangled polymer solution should be orders of magnitude greater than the residence time of the analyte molecules.
Unfortunately, this condition does not necessarily hold in typical CE applications, as demonstrated in the examples separating DNA by CE using entangled polymer solutions, reported by T. Hino, MS Thesis, University of California, Berkeley (1991), demonstrating that the slow-mode relaxation time of polyacrylamide was 5.9.times.10.sup.4 sec (T3%, 25.degree. C.) for entangled solutions and 4.0.times.10.sup.-3 sec (T3%, 25.degree. C.) for cross-linked systems. The typical residence time of DNA can be estimated from the literature (P. D. Grossman and D. S. Soane, Biopolymers, 31, 1221 (1991); P. D. Grossman and D. S. Soane, J. Chromatography, 559, 257 (1991); J. Sudor, F. Foret and P. Bocek, Electrophoresis, 12, 1056 (1991)) by assuming the two extremes (Ogston and biased reptation)=Residence time between ##EQU1## where subscript Ogston means that the migrating solute behaves as an undeformable particle (Ogston Model) and reptation under the influence of large electric fields, the solute becomes more elongated, and the motion mimics that of a snake threading its way through the network. The calculated residence time limits of DNA are as follows: 1.5.times.10.sup.-5 to 1.8.times.10.sup.-4 sec. for 30 base pairs and 1.5.times.10.sup.-5 to 8.2.times.10.sup.-4 sec. for 100 base pairs. The calculated residence times of 100 base pairs are extrapolated from 30 base pairs data based on a hydrodynamic diameter per base pair of 3.3 .ANG. (K. S. Schmitz, An Introduction to Dynamic Light Scattering by Macromolecules, Academic Press, NY (1990); V. A. Branfield, Chapter 10, Dynamic Light Scattering, Plenum Press, NY (1985)). The results show clearly that the relaxation time of entangled polymer and residence time of DNA are very close. Therefore, sharp resolution cannot be expected for the high molecular weight of DNA using entangled polymers. The network imposing the sieving medium fails before the analyte moves through a mesh completely.
In summary, the conventional electrophoretic methods are slow, labor-intensive, with relatively poorly reproducibility and have limited quantitative capability. Furthermore, it is difficult to accomplish a fully automated operation. CE promises to offer a solution for those problems. However, choosing the sieving medium is difficult. Open CE columns adsorb many proteins onto the walls of the capillaries. A cross-linked gel filled CE is complicated by problems in the routine preparation of homogenous stress-free gels in capillaries due to polymerization induced shrinkage and appearance of bubbles inside the capillaries. Entangled polymer solutions in the capillaries exhibit poor resolution and reproducibility. All the above problems can also be found with slab gel electrophoresis as well as other electrophoretic configurations.
It is therefore an object of the present invention to provide an improved method and medium composition for separation of molecules, especially by capillary gel electrophoresis.
It is a further object of the present invention to provide a method and means to improve the resolution and reproducibility of entangled solutions, in combination with the ease of fill/flush.
It is another object of the present invention to provide a new class of separation media for CE and related electrophoretic technologies, including slab and annular configurations, as well as sequencers for DNA and protein, and methods of preparation and use thereof.